PROJECT SUMMARY Accumulating evidence supports that regulatory B cells (Bregs), are essential for limiting inflammation and autoimmunity by IL-10-dependent and -independent manners. The lack of a universal marker for identifying Bregs, however, has hampered our understanding of their critical biologic functions. The processes and mechanisms by which Bregs are generated have also not been identified. Dr. Rothstein?s lab as well as our own have shown that Tim-1 identifies IL-10-producing Bregs. We have generated a mouse with loss of function Tim-1 mutant (Tim-1?mucin), and found that both Tim-1?mucin and traditional Tim-1 deficient (Tim-1-/-) show progressive loss of IL-10 production in B cells. Additionally, with age, these mice developed severe multi-organ tissue inflammation. We have recently demonstrated that Tim-1 on Bregs is required for apoptotic cell (AC) binding to Bregs and for AC-induced IL-10 production in Bregs. B cells from both mice are unable to produce IL-10 in response to AC. Thus, we hypothesis that Tim-1 is also critical and essential for optimal Breg function in maintaining immune tolerance and limiting inflammatory responses by sensing AC, which will be addressed in Aim 1 by using Tim-1 floxed mice we recently generated. In addition to producing IL-10, our RNA-seq analysis have demonstrated that Tim-1+ Bregs also express many other inhibitory molecules, some of which have been shown to regulate IL-10-independent Breg function. We have also demonstrated that some inhibitory molecules regulate IL-10 in Tim-1+ Bregs. Therefore, we hypothesize that Tim-1+ Bregs exert their regulatory function not only by producing IL-10, but also by expressing a panel of inhibitory molecules, which give Bregs the optimal ability to suppress autoimmune responses?this will be examined in Aim 2. Our RNA- seq dataset identified a set of transcription factors differentially expressed in Tim-1+ Bregs. Thus, we propose to study the role of the transcription factors in Bregs in Aim 3. These studies will greatly increase our understanding of the role of Tim-1 in Bregs and the role of Bregs in immune tolerance and autoimmunity, and may also provide a novel therapeutic strategy by targeting B cells for the treatment of autoimmune diseases. !